Cyanine-scaffold fluorogenic probes for visual detection of nitroreductase in living bacteria

Bacterial infections pose significant challenges in clinical diagnostics and microbiological research due to the need for rapid, sensitive, and specific detection methods. Herein, we report the development of Cy5-NO₂, a novel nitro-containing fluorescent probe designed for real-time monitoring of bacterial nitroreductase (NTR) activity. Cy5-NO₂ is synthesized through a streamlined, high-yield process without chromatography, yielding a stable compound confirmed by X-ray crystallography and spectroscopic methods. The probe exhibits negligible fluorescence in its native state but undergoes a 30-fold fluorescence enhancement at 620 nm upon NTR-mediated reduction of the nitro group to an amino group, with a detection limit of 10 ng mL⁻¹. Time-dependent density functional theory (TDDFT) calculations reveal that the fluorescence “turn-on” mechanism arises from a transition from charge-transfer quenching (in Cy5-NO₂) to local excitation (in Cy5-NH₂), as supported by a significant increase in oscillator strength. The probe demonstrates high specificity for NTR under physiological conditions and successfully detects live bacterial cells (e.g., E. coli and S. aureus) via confocal laser scanning microscopy (CLSM). The combination of direct nitro-to-amine reduction, exceptional stability, and minimal enzymatic interference positions Cy5-NO₂ as a promising tool for universal bacterial detection, advancing applications in clinical diagnostics and microbial imaging.